The long-term goal of this research project is to determine the biotin requirement for normal individuals and for individuals in specific clinical circumstances. Previous work conducted under this grant cast doubt on the analytic validity of a substantial proportion of reported studies of biotin nutriture: 1) Despite reports to the contrary, most of the biotin in human plasma is free in the aqueous phase. 2) Despite tactic assumptions to the contrary, substantial amounts of inactive biotin analogs are present in human plasma and urine. 3) Urinary excretion of these biotin analogs (e.g. biotin sulfoxide) is inducible by some drugs, including certain anticonvulsants; presumable, this phenomenon arises from induction of hepatic enzymes involved in the oxidation of biotin. 4) Urinary excretion of 3-hydroxyisovaleric acid (3-HIA), a leucine metabolite that is produced in response to reduced activity of the biotin-dependent enzyme methylcrotonyl-CoA carboxylase, appears to be an early and specific indicator of biotin depletion at the tissue level. Specific aims of this proposal are the following: 1) To determine the changes in the plasma concentrations and urinary excretion of biotin and its analogs during progression from biotin sufficiency to deficiency. This specific aim will test the hypothesis that the urinary excretion of biotin, measured by a chemically specific HPLC/avidin-binding assay for biotin and its analogs, will decrease to abnormally low values during frank deficiency in an animal model and during marginal biotin deficiency in normal adults, despite continued normal excretion rates of total avidin-binding substances. 2) To determine the usefulness of the urinary excretion of 3- HIA in detecting biotin deficiency under normal conditions and in response to a leucine challenge. This specific aim will test the hypothesis that urinary excretion of 3-HIA increases early in the course of marginal biotin deficiency in normal adults. Urinary excretion of 3-HIA will be determined by an improved gas chromatography/mass spectrometry method. 3) To determine the enzyme(s) responsible for biotin sulfoxidation. These experiments will initially investigate the possibility that the biotin sulfoxidase is a flavin monooxygenase or cytochrome P450 monooxygenase. 4) To determine whether biotin deficiency is associated with long-term therapy with certain anticonvulsants. This specific aim will test the hypothesis that a significant proportion of adults receiving long-term therapy with certain anticonvulsants become biotin deficient. Preliminary data indicate that treatment with carbamazepine and/or phenytoin causes biotin deficiency in children. 5) To determine whether biotin deficiency occurs during pregnancy. This study is particularly important because marginal biotin deficiency is a potent teratogen in several animal species. In Specific Aims 4 and 5, biotin status will be assessed by urinary excretion of 3-HIA, lymphocyte activity of the biotin-dependent enzyme propionyl-CoA carboxylase, plasma concentrations of biotin, and urinary excretion of biotin.